New insights are emerging into the role of CD1d and NKT cells in host defense against many microbial infections. In some of these infections, microbial lipid antigens are presented by CD1d and recognized by the semi-invariant T cell receptor (TCR) expressed by invariant (i)NKT cells. This is called direct, foreign antigen-driven iNKT cell activation. This is an important mechanism, but for many microbes that can activate iNKT cells it may not apply since they do not contain such cognate lipid antigens. For example, viruses, certain bacteria and fungi (as studied here) may not contain iNKT cell antigens. This led us to identify another mechanism of iNKT cell activation that is independent of recognition of microbial lipid antigens. Instead of lipid antigens, microbial TLR agonists stimulate antigen-presenting cells (APC) to secrete cytokines like IL-12 that synergize with weaker TCR stimulation provided by CD1d bound self-lipid antigens on the APC. These combined signals synergize to evoke strong iNKT cell activation, in the absence of iNKT cell TCR recognition of microbial lipid antigens. We call this mechanism indirect, cytokine-driven iNKT cell activation. Fungi cause a variety of important diseases, including local and systemic infections and mediate atopy in humans. We have found that several common fungal pathogens such as Candida albicans and Aspergillus fumigatus efficiently activate iNKT cells. Yet, we could identify no fungal lipid antigens that were recognized by iNKT cells. Moreover, fungi are different from bacteria as they contain poorly characterized TLR agonists. How fungi might activate iNKT cells and what role NKT cells play in control of major fungal infections in largely unknown. Here, our preliminary data reveal that fungi strikingly activate iNKT cells and appear to use a mechanism mediated by complex polysaccharides in their cells walls such as the characteristic 2-glucans, rather than fungal lipid antigens. Further, instead of TLR signaling, these fungal polysaccharides signal via the C-type lectin receptor, Dectin-1. Here we propose to define the molecular basis of iNKT cell activation by a panel of major fungi including A. fumigatus, C. albicans and C. neoformans in vitro (Aim 1) and in vivo (Aim 2) and then bring to light an unappreciated central role for iNKT cells in control of invasive fungal infection in vivo (Aim 3). Together, these studies will provide new insights into the how fungi active iNKT cells via their distinct polysaccharides and CD1d presented self-lipid antigens. Then we will determine how iNKT cells orchestrate the recruitment and activation of innate leukocytes like neutrophils and monocytes and impact the later adaptive CD4+ T cell response to control the fungal infection.